The RNeasy Fibrous Tissue Mini Kit includes proteinase K for removing abundant protein in fiber-rich tissue samples, and RNeasy spin columns for purifying up to 100 µg of high-quality RNA. The kit can be automated on theQIAcube. Fibrous tissue samples can be conveniently stabilized using RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor or TissueLyser system. For larger samples, the RNeasy Fibrous Tissue Midi Kit (spin-column binding capacity of 1 mg RNA) is also available.

RNA was isolated from the indicated rat tissues using the RNeasy Fibrous Tissue Mini Kit. Real-time, quantitative RT-PCR amplification was carried out with 100 ng total RNA using the QuantiTect Probe RT-PCR Kit and primers and probe specific for alpha-actin.

LightCycler analysis of high-quality RNA from heart.

RNA was isolated from mouse heart using the RNeasy Fibrous Tissue Mini Kit. After reverse transcription using the Omniscript RT Kit, real-time, quantitative, PCR was carried out on the LightCycler System using the QuantiTect SYBR® Green PCR Kit and primers specific for alpha-catenin. Amplification curves are shown for the indicated amounts of total RNA. NTC: no template control.

* RNA was isolated from the tissues indicated following kit suppliers' specifications.† Columns clogged, resulting in poor quality RNA. This yield may be overestimated due to contaminants in the prep.

Principle

RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification. RNeasy Fibrous Tissue Kits are optimized for use with fiber-rich tissues, such as skeletal muscle, heart, and aorta, tissues difficult to lyse due to the abundance of contractile proteins, connective tissue, and collagen. The RNeasy Fibrous Tissue protocol integrates proteinase K and RNase-free DNase digestion steps during RNA isolation to break down proteins and genomic DNA for higher yields of high-quality total RNA. Since the RNeasy procedures enrich for mRNA and other RNA species >200 nucleotides, the total RNA yield does not include 5S rRNA, tRNA, and other low-molecular-weight RNAs, which make up 15-20% of total cellular RNA.

Procedure

Samples of fibrous tissue from 0.5–30 mg are lysed in a guanidine-isothiocyanate buffer. After dilution of the lysate, the sample is treated with proteinase K. Debris is pelleted by centrifugation. Ethanol is then added to the cleared lysate, and RNA is bound to the RNeasy silica membrane. Traces of DNA that may copurify are removed by a DNase treatment on the RNeasy spin column. DNase and any contaminants are washed away, and up to 100 µg high-quality total RNA is eluted in 30–100 µl RNase-free water (see figure "RNeasy Fibrous Tissue Mini procedure").

Applications

RNeasy Fibrous Tissue Kits provide high-quality total RNA from fiber-rich samples that is suitable for any downstream application, including array analysis and real-time RT-PCR.